In Optics Letters, vol. 32, No. 14, Jul. 15, 2007, FIG. 1(c), Botcherby et al. describe a useful aberration-free internal focusing method which is schematically illustrated in FIG. 1 of the present description. O1 is the objective lens of the microscope, T1 is a first tube lens and T2 is a second tube lens.
On the side of a partially reflective beam splitter DM facing away from the objective lens O1, a plane mirror RM is disposed downstream of a second objective lens O2. The beam splitter couples out detection light from the sample in the direction of a detector.
The light emitted by the sample is collected by O1, and the 4f system that is formed by the tube lenses T1 and T2 forms pupil P1 of the first objective lens into pupil P2 of the second objective lens. As a result, the second objective lens O2 which is identical to the first objective lens O1 generates a three-dimensional image of the sample in its focal plane.
A suitable detector that would lie in the focal plane of O2 could detect a diffraction-limited image of a selected image region.
The plane mirror RM is disposed in the focal plane of O2 and can be moved relative to the optical axis.
The optical system comprising O2, the beam splitter DM, a tube lens T3 and the detector generates an image of the focal plane of O2 in the plane of the detector. By moving mirror RM along the optical axis, the plane of interest of the sample is imaged into the plane of the detector. By back-imaging through objective lens O2 which is identical to O1, the spherical aberrations generated by the optical system used are compensated for, and an aberration-free diffraction-limited image is generated in the plane of the detector.
Using a partially reflective beam splitter as shown in FIG. 1 entails light losses of up to 75% during the detection of the sample light, and as a result, the sensitivity of the system is reduced. Especially when working with weakly fluorescing samples, this can be a marked disadvantage.